Yushu Joy Xie

Significance Despite its success in treating hematological cancers, chimeric antigen receptor (CAR) T cell therapy does not so easily eliminate solid tumors. Solid tumors generally develop in a highly immunosuppressive environment and are difficult to target, mostly due to a lack of tumor-specific antigen expression, but other factors contribute as well. This study develops a strategy to target multiple solid tumor types through markers in their microenvironment. The use of single-domain antibody (VHH)-based chimeric antigen receptor (CAR) T cells that recognize these markers circumvents the need for tumor-specific targets. VHH-based CAR T cells that target the tumor microenvironment through immune checkpoint receptors or through stroma and ECM markers are effective against solid tumors in syngeneic, immunocompetent animal models.

An approach to proteomic analysis that combines bioorthogonal noncanonical amino acid tagging (BONCAT) and pulsed stable isotope labeling with amino acids in cell culture (pSILAC) provides accurate quantitative information about rates of cellular protein synthesis on time scales of minutes. The method is capable of quantifying 1400 proteins produced by HeLa cells during a 30 min interval, a time scale that is inaccessible to isotope labeling techniques alone. Potential artifacts in protein quantification can be reduced to insignificant levels by limiting the extent of noncanonical amino acid tagging. We find no evidence for artifacts in protein identification in experiments that combine the BONCAT and pSILAC methods. An approach to proteomic analysis that combines bioorthogonal noncanonical amino acid tagging (BONCAT) and pulsed stable isotope labeling with amino acids in cell culture (pSILAC) provides accurate quantitative information about rates of cellular protein synthesis on time scales of minutes. The method is capable of quantifying 1400 proteins produced by HeLa cells during a 30 min interval, a time scale that is inaccessible to isotope labeling techniques alone. Potential artifacts in protein quantification can be reduced to insignificant levels by limiting the extent of noncanonical amino acid tagging. We find no evidence for artifacts in protein identification in experiments that combine the BONCAT and pSILAC methods. Methods for the analysis of cellular protein synthesis should be quantitative and fast. In 2006, Dieterich and coworkers introduced a proteomics discovery tool called bioorthogonal noncanonical amino acid tagging (BONCAT), 1The abbreviations used are: BONCAT, Bioorthogonal noncanonical amino acid tagging; pSILAC, Pulsed stable isotope labeling with amino acids in cell culture; Aha, L-azidohomoalanine; Aha30:1, A mixture of Aha (1 mM) and Met (33 μM); ncAA, Noncanonical amino acid; H/M, Heavy to medium ratio; GO, Gene ontology. 1The abbreviations used are: BONCAT, Bioorthogonal noncanonical amino acid tagging; pSILAC, Pulsed stable isotope labeling with amino acids in cell culture; Aha, L-azidohomoalanine; Aha30:1, A mixture of Aha (1 mM) and Met (33 μM); ncAA, Noncanonical amino acid; H/M, Heavy to medium ratio; GO, Gene ontology. in which noncanonical amino acids (ncAAs) with bioorthogonal functional groups (e.g. azides or alkynes) are used as metabolic labels to distinguish new proteins from old (1.Dieterich D.C. Link A.J. Graumann J. Tirrell D.A. Schuman E.M. Selective identification of newly synthesized proteins in mammalian cells using bioorthogonal noncanonical amino acid tagging (BONCAT).Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 9482-9487Crossref PubMed Scopus (551) Google Scholar, 2.Dieterich D.C. Lee J.J. Link A.J. Graumann J. Tirrell D.A. Schuman E.M. Labeling, detection and identification of newly synthesized proteomes with bioorthogonal non-canonical amino-acid tagging.Nat. Protoc. 2007; 2: 532-540Crossref PubMed Scopus (227) Google Scholar). Labeled proteins can be conjugated to fluorescent reporters for visualization or affinity tags for purification and subsequent identification by mass spectrometry (3.Szychowski J. Mahdavi A. Hodas J.J.L. Bagert J.D. Ngo J.T. Landgraf P. Dieterich D.C. Schuman E.M. Tirrell D.A. Cleavable biotin probes for labeling of biomolecules via azide-alkyne cycloaddition.J. Am. Chem. Soc. 2010; 132: 18351-18360Crossref PubMed Scopus (147) Google Scholar). Because the ncAA probe can be introduced to cells in a well-defined “pulse,” affinity purification removes pre-existing proteins and provides both reduced sample complexity and excellent time resolution. The methionine (Met) surrogate l-azidohomoalanine (Aha) has become standard in the application of BONCAT methodologies. Using Aha and fluorescent tagging, Tcherkezian et al. observed co-localization of the DCC receptor with sites of protein synthesis, providing support for the role of netrin as a stimulant of extranuclear protein production in neurons (4.Tcherkezian J. Brittis P.A. Thomas F. Roux P.P. Flanagan J.G. Transmembrane receptor DCC associates with protein synthesis machinery and regulates translation.Cell. 2010; 141: 632-644Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). Combining Aha labeling and 2D gel electrophoresis, Yoon et al. discovered that the protein lamin B2 is synthesized in axons and crucial to mitochondrial function and axon maintenance in Xenopus retinal glial cells (5.Yoon B.C. Jung H. Dwivedy A. Hare C.M.O. Zivraj K.H. Holt C.E. Local translation of extranuclear lamin B promotes axon maintenance.Cell. 2011; 148: 752-764Abstract Full Text Full Text PDF Scopus (200) Google Scholar). Aha has also been used to study histone turnover (6.Deal R.B. Henikoff J.G. Henikoff S. Genome-wide kinetics of nucleosome turnover determined by metabolic labeling of histones.Science. 2010; 328: 1161-1164Crossref PubMed Scopus (362) Google Scholar), protein palmitoylation (7.Zhang M.M. Tsou L.K. Charron G. Raghavan A.S. Hang H.C. Tandem fluorescence imaging of dynamic S-acylation and protein turnover.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 8627-8632Crossref PubMed Scopus (87) Google Scholar), pathogen amino acid uptake (8.Ouellette S.P. Dorsey F.C. Moshiach S. Cleveland J.L. Carabeo R.A. Chlamydia species-dependent differences in the growth requirement for lysosomes.PLoS One. 2011; 6: e16783Crossref PubMed Scopus (56) Google Scholar), inflammatory response (9.Choi K.-Y.G. Lippert D.N.D. Ezzatti P. Mookherjee N. Defining TNF-α and IL-1β induced nascent proteins: Combining bio-orthogonal non-canonical amino acid tagging and proteomics.J. Immunol. Methods. 2012; 382: 189-195Crossref PubMed Scopus (12) Google Scholar), and local translation in neuronal dendrites and axons (10.Melemedjian O.K. Asiedu M.N. Tillu D.V. Peebles K.A. Yan J. Ertz N. Dussor G.O. Price T.J. IL-6- and NGF-induced rapid control of protein synthesis and nociceptive plasticity via convergent signaling to the eIF4F complex.J. Neurosci. 2010; 30: 15113-15123Crossref PubMed Scopus (162) Google Scholar). These labeling techniques have been expanded to tissue and animal culture, where Aha has been used to profile protein synthesis in rat hippocampal brain slices (11.Dieterich D.C. Hodas J.J.L. Gouzer G. Shadrin I.Y. Ngo J.T. Triller A. Tirrell D.A. Schuman E.M. In situ visualization and dynamics of newly synthesized proteins in rat hippocampal neurons.Nat. Neurosci. 2010; 13: 897-905Crossref PubMed Scopus (317) Google Scholar, 12.Hodas J.J.L. Nehring A. Höche N. Sweredoski M.J. Pielot R. Hess S. Tirrell D.A. Dieterich D.C. Schuman E.M. Dopaminergic modulation of the hippocampal neuropil proteome identified by bio-orthogonal non-canonical amino-acid tagging (BONCAT).Proteomics. 2012; 12: 2464-2476Crossref PubMed Scopus (52) Google Scholar) and zebrafish embryos (13.Hinz F.I. Dieterich D.C. Tirrell D.A. Schuman E.M. Noncanonical amino acid labeling in vivo to visualize and affinity purify newly synthesized proteins in larval zebrafish.ACS Chem. Neurosci. 2012; 3: 40-49Crossref PubMed Scopus (91) Google Scholar). The development of fast, reliable, quantitative BONCAT methods will enable new insights into proteome dynamics in response to biological stimuli. Recent work by Eichelbaum et al. combined Aha labeling with stable isotope labeling to measure lipopolysaccharide-stimulated protein secretion by macrophages (14.Eichelbaum K. Winter M. Diaz M.B. Herzig S. Krijgsveld J. Selective enrichment of newly synthesized proteins for quantitative secretome analysis.Nat. Biotechnol. 2012; 30: 984-990Crossref PubMed Scopus (185) Google Scholar). Using similar approaches, Somasekharan et al. identified a set of proteins that are translationally regulated by the Y-box binding protein-1 (YB-1) in TC-32 Ewing sarcoma cells (15.Somasekharan S.P. Stoynov N. Rotblat B. Leprivier G. Galpin J.D. Ahern C. a Foster L.J. Sorensen P.H.B. Identification and quantification of newly synthesized proteins translationally regulated by YB-1 using a novel Click-SILAC approach.J. Proteomics. 2012; 77: e1-e10Crossref PubMed Scopus (37) Google Scholar), and Howden et al. monitored changes in protein expression following stimulation of primary T cells with phorbol 12-myristate 13-acetate and ionomycin (16.Howden A.J.M. Geoghegan V. Katsch K. Efstathiou G. Bhushan B. Boutureira O. Thomas B. Trudgian D.C. Kessler B.M. Dieterich D.C. Davis B.G. Acuto O. QuaNCAT: quantitating proteome dynamics in primary cells.Nat. Methods. 2013; 10: 343-346Crossref PubMed Scopus (120) Google Scholar). A concern that arises in the use of Aha (as it does for all chemical probes of biological processes) is that the protocols used for Aha labeling might perturb cellular protein synthesis. The development of ncAAs as reliable analytic tools hinges on our ability to understand and minimize such unintended effects. For Aha, previous work has shown that protein labeling does not visibly alter cellular morphology in dissociated hippocampal neurons or HEK293 cells, and 1D gels reveal no discrepancies between the proteomes of Aha- and Met-treated cells (1.Dieterich D.C. Link A.J. Graumann J. Tirrell D.A. Schuman E.M. Selective identification of newly synthesized proteins in mammalian cells using bioorthogonal noncanonical amino acid tagging (BONCAT).Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 9482-9487Crossref PubMed Scopus (551) Google Scholar). These experiments, however, offer only coarse measures of effects on protein synthesis, and as Aha labeling is frequently coupled to mass spectrometry-based proteomic analysis, the biological effects of Aha treatment must be investigated with equivalent sensitivity and resolution. Here we report sound methods for fast, reliable measurement of proteome dynamics via noncanonical amino acid tagging. First, we use the quantitative proteomics technique pulsed stable isotope labeling with amino acids in cell culture (pSILAC) to investigate potential unintended effects of Aha labeling on protein abundance in HeLa cell cultures, and we develop a strategy for minimizing these effects. Second, we show that a combined BONCAT-pSILAC approach, capable of both enriching and quantifying newly synthesized proteins, yields detailed proteomic information on time scales that are inaccessible to isotope labeling techniques alone. HeLa cells were maintained in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% FBS (Invitrogen) and 1% penicillin/streptomycin (Invitrogen) in a humidified incubator at 37 °C and 5% CO2. For each pSILAC experiment, 2.1 million cells were seeded in 2 T-75 flasks and grown for 24 h. Cultures were washed with warm PBS twice and resuspended in custom lysine-free and Met-free DMEM (Invitrogen) supplemented with either “medium” lysine (D4 l-lysine, Cambridge Isotope Laboratories) or “heavy” lysine (U-13C6 U-15N2 l-lysine, Cambridge Isotope Laboratories, Andover, MA) at 1 mm. Cultures were also supplemented with either Met (1 mm), Aha (1 mm), or Aha30:1 (1 mm Aha, 33 μm Met) as indicated for each experiment. Aha was synthesized as previously described (17.Link A.J. Vink M.K.S. Tirrell D.A. Synthesis of the functionalizable methionine surrogate azidohomoalanine using Boc-homoserine as precursor.Nat. Protoc. 2007; 2: 1884-1887Crossref PubMed Scopus (10) Google Scholar). pSILAC experiments measuring changes in protein abundance upon treatment with Aha or Aha30:1 and Met were conducted with four biological replicates, two of which were arranged as label swap experiments. pSILAC experiments with pulse durations of 4 h and 30 min were performed with three biological replicates. After the desired labeling time, cells were removed from the flask by trypsinization and pelleted at 4 °C. Cells were lysed in 2% SDS in PBS by heating to 90 °C for 10 min. DNA was digested with Benzonase (Sigma) and lysates were cleared by centrifugation. Protein concentrations were measured with the BCA protein quantitation kit (Thermo Scientific). BONCAT experiments were carried out as described in the HeLa cell pSILAC protocol, with a few modifications. T-150 flasks were seeded with 4 million cells prior to each experiment. The larger culture size compensates for the relatively small amounts of protein that are produced during short pulses. During the pulse, both medium and heavy cultures were supplemented with either Aha or Aha30:1. Each BONCAT experiment was conducted with three biological replicates. Protein synthesis was halted prior to cell lysis by addition of cycloheximide (Sigma) to 100 μg/ml. Cells were lysed in freshly prepared 2% SDS in PBS with 100 mm chloroacetamide (Sigma) to alkylate free cysteines in proteins. Cysteine alkylation reduces thiol addition of cyclooctyne reagents, and increases the specificity of tagging of Aha-labeled proteins (18.Van Geel R. Pruijn G.J.M. van Delft F.L. Boelens W.C. Preventing thiol-yne addition improves the specificity of strain-promoted azide-alkyne cycloaddition.Bioconjugate Chem. 2012; 23: 392-398Crossref PubMed Scopus (204) Google Scholar). After mixing heavy and medium lysates, Aha-labeled proteins were conjugated to a biotin tag by strain-promoted azide-alkyne click chemistry (19.Debets M.F. van Berkel S.S. Schoffelen S. Rutjes F.P. J.T. van Hest J.C.M. van Delft F.L. Aza-dibenzocyclooctynes for fast and efficient enzyme PEGylation via copper-free (3+2) cycloaddition.Chem. Commun. 2010; 46: 97-99Crossref PubMed Scopus (440) Google Scholar). DBCO-sulfo-biotin tag (Click Chemistry Tools (Scottsdale, AZ)) was added to 1 mg of mixed lysates to a final concentration of 12 μm and allowed to react for 15 min, after which the reaction was quenched with excess Aha. Tagged proteins were with (Thermo washed with of 1% SDS in and by the in 1 mm biotin in 1% SDS in PBS for 15 min. proteins were on a MA) prior to of excess or biotin tag prior to was of the small of tag used in the click were on gels (Invitrogen) and with the kit were into gel and by with mm and mm and were reduced in mm in mm at °C for 30 min. After the proteins were with freshly prepared mm chloroacetamide (Sigma) in mm for min in the were washed with mm and for min were digested with at in mm at 37 °C. were from gel by in 1% for min, for min, and 1% acid in for min. were with custom as described in et al. J. M. for and of for proteomics using Protoc. 2007; 2: PubMed Scopus Google Scholar), by and resuspended in acid (Sigma) prior to spectrometry experiments were performed on MA) to a or (Thermo with a Scientific). pSILAC experiments with 24 h pulse were on the and experiments pSILAC and BONCAT with 4 h and 30 min pulse were on the on the were on a 15 μm with μm using a min from to B at a of A was 2% and B was and For the a 90 min from to B was The mass were in to between and as described A. 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In pSILAC experiments Met Aha and Aha30:1 Met-treated cultures, we report only proteins in both of the label swap experiments, and that Met were protein to of protein and standard were using a combined with and at the a of measurement is using from the protein in each a of the protein is by the protein as the of the in each and the protein as the of the protein the standard of the protein is using a where with the at both the and and each in the is by a from a with and standard to the previously of measurement In are The standard of the protein is as the standard of the protein can be used to of protein with to a In pSILAC, metabolic labeling with amino acid is used to rates of protein translation in cell B. M. G. M. analysis of cellular protein translation by pulsed PubMed Scopus Google Scholar). measure the of Aha treatment on protein cells grown in standard were and into “medium” and “heavy” culture either Aha, or a mixture of Aha and Met After labeling of 24 h in HeLa cell culture, medium and heavy cultures were lysed and mixed in a experiments in which both medium and heavy were with Met as were prepared for by standard gel and were with the quantitative proteomics J. M. identification mass and protein Biotechnol. PubMed Scopus Google Scholar). The extent of of Met by Aha can be by the concentrations of the two amino acids in the culture medium C. G. Tirrell D.A. of proteins: and by Scopus Google Scholar). 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These that Met of Aha to the changes in protein abundance observed in The extent to which the observed changes are of Aha Met or a of the however, is A similar of the of labeling of HeLa cells with a mixture of Aha and Met that labeling yields reliable of protein The of proteins from Aha30:1 and cultures and quantitative are in Because BONCAT provides a of newly synthesized cellular proteins, we the pSILAC experiments for evidence that Aha labeling might to artifacts in protein medium and heavy lysine labels allowed for of proteins identified in either Aha- or cultures from pSILAC experiments. For each experiment we determined the of proteins the between the sample with either Aha or Aha30:1 and the Met and the of proteins only in the sample culture In each the of proteins only in the sample culture between and of the and no was observed in cultures with with Met we find no evidence that Aha of Met to artifacts in protein BONCAT and pSILAC can be used to measure changes in the cellular proteome that during time by amino acid The between the two methods is that BONCAT newly synthesized proteins prior to analysis, pSILAC and new proteins the BONCAT and pSILAC approaches, newly synthesized proteins produced during a pulse can be both and Here we show that quantitative BONCAT can be used as a approach for rapid proteomic and we the of BONCAT and pSILAC for short labeling For short newly synthesized proteins a small of the We that enriching newly synthesized proteins by with Aha in detection and quantification by mass spectrometry for short pulse We the of the combined BONCAT-pSILAC approach to that of pSILAC at pulse of 4 h and 30 min in HeLa cell In BONCAT experiments, Aha-labeled proteins were by to a tag via strain-promoted azide-alkyne click chemistry and purification on a E.M. Bioorthogonal Chem. 2011; PubMed Scopus Google Scholar). proteins were prepared for as described in the methods. Using the combined BONCAT-pSILAC method in experiments, we and newly synthesized proteins in HeLa cell cultures a 4 h with the Aha and Aha30:1 labeling pSILAC experiments proteins the 4 h pulse For a pulse time of 30 min, pSILAC only newly synthesized proteins, BONCAT and proteins with the Aha and Aha30:1 labeling of protein produced similar BONCAT-pSILAC experiments protein that were accurate and about a of 1 BONCAT were of of proteins between BONCAT experiments pSILAC experiments in the 4 h pulse, and in the 30 pulse experiments to newly synthesized proteins in the 4 h and 30 min experiments and of the After BONCAT newly synthesized proteins of and of the protein in the 4 h and 30 min experiments, The of proteins from the 4 h and 30 min pSILAC and BONCAT experiments is in with Aha fast, accurate and detection of changes in the cellular of Met by Aha, for of 24 h in HeLa cell culture, does not the of proteins by mass differences in protein abundance are observed such the we Aha30:1 labeling strategy that in protein abundance a of labeling that is for by click chemistry and affinity The Aha30:1 labeling approach is for use in in which of protein is of the Aha labeling increases the of affinity enrichment and can be used to larger of newly synthesized proteins, in which enrichment is as in experiments that use short pulse A of the BONCAT and pSILAC methods that affinity enrichment of newly synthesized proteins the time of proteomic A combined BONCAT-pSILAC approach of quantitative proteomic information 30 min in HeLa cell culture, a time scale that is inaccessible to isotope labeling techniques alone. 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Chimeric antigen receptor (CAR) T-cell therapy is effective in the treatment of cancers of hematopoietic origin. In the immunosuppressive solid tumor environment, CAR T cells encounter obstacles that compromise their efficacy. We developed a strategy to address these barriers by having CAR T cells secrete single-domain antibody fragments [variable heavy domain of heavy chain antibodies (VHH) or nanobodies] that can modify the intratumoral immune landscape and thus support CAR T-cell function in immunocompetent animals. VHHs are small in size and able to avoid domain swapping when multiple nanobodies are expressed simultaneously-features that can endow CAR T cells with desirable properties. The secretion of an anti-CD47 VHH by CAR T cells improves engagement of the innate immune system, enables epitope spreading, and can enhance the antitumor response. CAR T cells that secrete anti-PD-L1 or anti-CTLA-4 nanobodies show improved persistence and demonstrate the versatility of this approach. Furthermore, local delivery of secreted anti-CD47 VHH-Fc fusions by CAR T cells at the tumor site limits their systemic toxicity. CAR T cells can be further engineered to simultaneously secrete multiple modalities, allowing for even greater tailoring of the antitumor immune response.

T cell responses to diverse epitopes in animal models, alone or when co-administered with BNT162b2 while preserving spike-specific immunity. Importantly, we demonstrate that BNT162b4 protects hamsters from severe disease and reduces viral titers following challenge with viral variants. These data suggest that a combination of BNT162b2 and BNT162b4 could reduce COVID-19 disease severity and duration caused by circulating or future variants. BNT162b4 is currently being clinically evaluated in combination with the BA.4/BA.5 Omicron-updated bivalent BNT162b2 (NCT05541861).